The objective of the grant proposal is to define the mechanism by which hormones and other cell stimuli impose control over the enzyme activity of phospholipase C which is believed to be the initial enzyme catalyzed step in the two step reaction sequence involved in the release of archidonic acid from membrane phospholipids. Arachidonic acid, when released, undergoes conversion to metabolites believed to serve as important regulators of cell function. Phospholipase C activity will be examined with respect to its regulation by membrane related components and enzyme-promoted interconversions. Preliminary experiments indicate that the majority of phospholipase C activity resides in the soluble fraction of human platelets and that calcium ions (Ka 5-10 micrometers required for enzyme activity. It was also discovered that phospholipase C activity could be markedly inhibited by a cyclic AMP promoted event in intact platelets. This observation suggests that phospholipase C is a site of action of inhibitors of platelet aggregation such as prostacyclin. The proposed studies will include the investigation of the possible involvement of Ca-dependent regulator (CDR) in relation to the calcium-dependence of phospholipase C activity and the effects inducible on phospholipase C activity by phosphorylation catalyzed cyclic AMP and Ca2 ion CDR dependent protein kinases, dephosphorylation, and oxidation by substances such as prostaglandin endoperoxides and specific fatty acid hydroperoxides. Experiments will also be conducted to examine whether Ca ion and/or various membrane phospholipids may regulate phospholipase C activity by altering the binding of the enzyme to phosphatidylinositol, an obligatory step in this catalysis. The effects of these membrane components and enzyme-promoted alterations will be investigated using phospholipase C activity of human platelets in situ, in subcellular fractions of cell lysates and a purified form of the soluble enzyme.